Previous studies have shown considerable variability in the metabolic response of human skeletal muscle during a standardized exercise protocol. The goal of these studies was to investigate the factors responsible for the broad range of metabolic changes produced by fatiguing exercise. Experiments were performed to quantitate the measurement error of 31P nuclear magnetic resonance spectroscopy of human muscle, the reproducibility of changes within a single subject, and the effects of fasting, carbohydrate loading, and metabolic acidosis. The results show that none of these factors appear to be responsible for the wide variation between subjects. However, the effects of training and genetic factors were not investigated and are likely to be responsible for the substantial variability between subjects.